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Structural Study of Interaction Between the FMN Binding Domain of Cytochrome P450 Reductase and Its Redox Partners

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Structural Study of Interaction Between the FMN Binding Domain of Cytochrome P450 Reductase and Its Redox Partners Structural Study of Interaction between the FMN Binding Domain of Cytochrome P450 Reductase and Its Redox Partners – Cytochrome P450/Cytochrome c by NMR and NMR Characterization of Monomeric and Oligomeric Conformations of Human Calcitonin and Its Interaction with EGCG by Rui Huang A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Chemistry) in the University of Michigan 2014 Doctoral Committee: Professor Ayyalusamy Ramamoorthy, Chair Professor Zhan Chen Assistant Professor Kenichi Kuroda Associate Professor Michal R. Zochowski Acknowledgements I would like to first thank my supervisor, Dr. A. Ramamoorthy, for his constant support along the past five years. His advice and guidance has been very valuable not only for the progress of my project but also for my scientific career in general. In particular, his encouragement for innovation and his willingness to try the impossible has inspired me and going to continue to affect me for my future career. I would also like to thank my committee members, Dr. Chen, Dr. Kuroda and Dr. Zochowski. Your support and suggestions on my project have been very helpful to me. A great many thanks should go to the members of the Rams lab including the ones that have gone and the new ones that I have just fortunately met. To Shivani, Nataliya and Vivek, you have taught me most of the things that I need to know for my project. To Sam, Meng, Manoj, Neil, and Kamal, I really cherish our friendship, and thank you for being there along the way. And to Patrick, I wish I could have known you much earlier and I feel very lucky to meet you in my final year. And for the rest of the lab, thank you for always being so helpful and encouraging. You are the ones who make the lab not only a working environment for me, but a place that I belong to. Special thanks go to my great parents, who have always encouraged me to peruse my dreams. Thank you for always standing behind me even being thousands of miles away. Thank you for making me the person who I am. Thank you, my C222 family in Ann Abor. To Laoge, Chengzi, Angelica and Suchandan, I cannot imagine my life without you in these five years. I feel very grateful that we came together at this time of my life, and those memories we had will be accompany me lifelong. Finally, I’m most grateful to my love, Juan Felipe, for standing beside me in good and bad times. I could not have done this without you. Thank you. ii Table of Contents Acknowledgements ......................................................................................................................... ii List of tables ................................................................................................................................ viii List of figures ................................................................................................................................ ix List of appendices ....................................................................................................................... xiii List of abbreviations ................................................................................................................... xiv Abstract ....................................................................................................................................... xvi Chapter 1 Introduction .....................................................................................................................1 1.1 NADPH-cytochrome P450 reductase ...................................................................................1 1.1.1 Background and significance ........................................................................................1 1.1.2 Structural characteristics of CPR ...................................................................................2 1.1.3 Mechanism of internal electron transfer in CPR ............................................................6 1.1.4 Domain movement of CPR ...........................................................................................9 1.2 Cytochrome P450 ................................................................................................................11 1.2.1 Background and significance .......................................................................................11 1.2.2 Structure of cytochrome P450 .....................................................................................12 1.3 Interaction and electron transfer between CPR and cyt P450 ............................................14 1.4 Interaction and electron transfer between CPR and cytochrome c ....................................17 1.5 Goal of this research ...........................................................................................................20 1.6 Experimental methods for studying interaction and electron transfer between proteins ...21 1.6.1 Solution NMR experiments .........................................................................................21 1.6.2 High Ambiguity Driven biomolecular DOCKing (HADDOCK) ...............................23 1.6.3 Electron transfer pathway prediction ...........................................................................24 1.6.4 Kinetics studies by stopped-flow spectroscopy ...........................................................25 iii 1.6.5 Solid-state NMR experiments .....................................................................................26 1.7 References ..........................................................................................................................30 Chapter 2 Characterization of Interaction and Electron Transfer between the FMN Binding Domain of Cytochrome P450 Reductase and Cytochrome c .....................................................39 2.1 Summary ............................................................................................................................39 2.2 Introduction ........................................................................................................................39 2.3 Materials and methods .......................................................................................................42 2.3.1 Materials ......................................................................................................................42 2.3.2 Expression and purification of a uniformly 15N-labeled FMN binding domain of rat CPR ..............................................................................................................................42 2.3.3 Titration of FBD by dithionite under anaerobic conditions ........................................43 2.3.4 Pre-steady-state kinetics of cyt c reduction by FBD/CPR ...........................................43 2.3.5 Kinetics of auto-oxidation of hydroquinone FBD .......................................................44 2.3.6 NMR titration experiments ..........................................................................................44 2.3.7 Structural modeling of FBD-cyt c complex ................................................................45 2.4 Results ................................................................................................................................45 2.4.1 Characterization of the FMN binding domain .............................................................45 2.4.2 Kinetics studies of electron transfer between the FMN binding domain and cytochrome c in comparison with wild-type CPR ........................................................46 2.4.3 Determination of dissociation constant of FBD and cyt c by NMR titrations ............50 2.4.4 Chemical shift mapping of binding interface on FBD ................................................54 2.4.5 Structural model of FBD-cyt c complex .....................................................................57 2.5 Discussion ..........................................................................................................................60 2.5.1 Electron transfer between FBD and cyt c in comparison with CPR ...........................60 2.5.2 Complex formation between FBD and cyt c ...............................................................61 2.6 Conclusion ..........................................................................................................................64 iv 2.7 References ..........................................................................................................................65 Chapter 3 Characterization of Electron Transfer and Interaction between the FMN binding domain of Cytochrome P450 Reductase and Cytochrome P450 ...............................................70 3.1 Summary ............................................................................................................................70 3.2 Introduction ........................................................................................................................70 3.3 Materials and methods .......................................................................................................72 3.3.1 Materials ......................................................................................................................72 3.3.2 Expression and purification of proteins .......................................................................72 3.3.3 Pre-steady-state kinetics of cyt P450 reduction by FBD/CPR ....................................73 3.3.4 NMR titration experiments ..........................................................................................73
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